cgrp fragment 8–37  (Millipore)

Journal: The journal of headache and pain

Article Title: CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine.

doi: 10.1186/s10194-022-01531-8

Figure Lengend Snippet: Fig. 1 Chronic NTG injection evoked sustained CM-like phenotypes. A Schematic illustration of chronic NTG injection protocol. Mice of both sexes were i.p. injected with either vehicle (Ctrl) or NTG (0.1 mg/kg). B On day 1, the basal mechanical threshold was the same between the NTG and control group, while the formal developed mechanical hyperalgesia after 2 hrs post-treatment (left, n = 18 per group; p < 0.01 **, and p < 0.001 ***). Sustained basal mechanical hyperalgesia developed after repeated doses of NTG administration (right, n = 18 per group; p < 0.001 ***). C The basal mechanical threshold recovered 6 days after the final NTG injection (n = 4 per group; F(1,24) = 16.2; p < 0.05 *, and p < 0.01 **). D Schematic illustration of periorbital test protocol. The periorbital responses were scored 2 hrs after the i.p. injection of the NTG (left). Sustained mechanical hyperalgesia developed after repeated doses of NTG administration in both sexes of mice (right, n = 9 per group; F(1,112) = 117.8; p < 0.05 *, p < 0.01 **, and p < 0.001 ***). E Sustained mechanical hyperalgesia developed after repeated doses of NTG administration in male mice (n = 5 per group; F(1,4) = 71.6; p < 0.05 *, and p < 0.001 ***). F Sustained mechanical hyperalgesia developed after repeated doses of NTG administration in female mice (n = 4 per group; p < 0.05 *, and p < 0.01 **). G Schematic illustration of acute sumatriptan treatment protocol. Mice of both sexes were i.p. injected with sumatriptan (0.6 mg/kg) 5 min after NTG injection on day 9 (left). Acute sumatriptan injection transiently alleviated chronic NTG induced mechanical hyperalgesia on day 9. The basal response in the NTG group returned to mechanical hyperalgesia on day 10, suggesting that acute sumatriptan treatment only aborted acute pain but did not reverse the chronicity of mechanical hyperalgesia. (right, n = 20 per group; p < 0.001 ***). H Acute sumatriptan injection transiently alleviated chronic NTG induced mechanical hyperalgesia in male mice (n = 10 per group; p < 0.05 *, and p < 0.01 **). I Acute sumatriptan injection transiently alleviated chronic NTG induced mechanical hyperalgesia in female mice (n = 10 per group, p < 0.001 ***). J Schematic illustration of chronic NTG injection protocol. Mice were sacrificed after the NTG post-treatment von Frey test on day 9. K Representative images show that co-labeled CGRP and pERK neurons in the TG after chronic NTG administration were significantly higher than that in the controls (right, n = 4 per group; p = 0.043). Scale bar: 200 μm. l Quantification of the coloalized percentage of CGRP and pERK-positive neurons in the TG after chronic NTG induction (n = 4 per group; p = 0.003). All data shown are mean ± SEM and analyzed by Friedman tests with Dunn’s post hoc test (B, F, G-I) or Bonferroni post hoc test (C-E) or Mann-Whitney U test (K, L). Significance levels set at p < 0.05 *, p < 0.01 **, and p < 0.001 ***

Article Snippet: Stereotactic or cannula microinfusion of calcitonin gene‐related peptide receptor antagonist into CeA To block the PBN CGRP neurotransmission to the CeA, we applied the CGRP receptor 1 antagonist, CGRP fragment 8–37 (HY-P0209, MedChemExpress), dissolved in normal saline at 1.8 μg/μl [19], into bilateral CeA with either acute stereotactic microinfusion or intermittent infusion via chronically implanted cannulas.

Techniques: Injection, Control, Labeling, MANN-WHITNEY

Journal: The journal of headache and pain

Article Title: CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine.

doi: 10.1186/s10194-022-01531-8

Figure Lengend Snippet: Fig. 3 Expression of CGRP after chronic NTG administration. A Schematic illustration of the rostro-caudal anatomical location of CeA relative to the position of bregma. B Representative images of CGRP expression in the CeA after chronic NTG administration. Red dashed square indicates the high magnification of the CeA (left). The CGRP fiber intensity in the CeA after chronic NTG induction was significantly higher than that in the control group (right, n = 4 per group; CeA_R; p = 0.003; CeA_L; p = 0.0003). Scale bar: 1 mm (left), 50 μm (right). C The representative pERK positive neurons double-labeled with CGRP fibers. Scale bar: 100 μm. D Representative low and high magnification images of co-labeled CGRP receptor type 1 (CGRPR)- and PKC-δ- positive neurons in the CeA (left) and quantified results (right, n = 4 per group; CGRPR/PKC-δ; p = 0.593; PKC-δ/CGRPR; p = 0.687). Scale bar: 50 μm. E Representative low magnification images of co-labeled CGRP- and pERK- positive neurons in the PBN. Scale bar: 500 μm. F Representative low and high magnification images of co-labeled CGRP- and pERK- positive neurons in the PBN (left). The CGRP positive neurons in the PBN was significantly higher after chronic NTG administration than controls (right, n = 4 per group; CGRP/pERK; p < 0.0001; pERK/ CGRP; p = 0.002). Scale bar: 100 μm. G Representative images of bilateral retrograde CTB594 labeling and co-labeled CGRP fibers in the CeA. Scale bar: 100 μm. H Representative images of CTB594 co-labeled CGRP positive neurons in the PBN, confirming that the CGRP positive neurons project from the PBN to CeA (left). The CGRP positive neurons in the PBN was significantly higher after chronic NTG administration than controls (right, n = 4 per group; CTB594/CGRP; p = 0.002; CGRP/CTB594; p = 0.0006). Scale bar: 100 μm. All data shown are mean ± SEM and analyzed by Mann-Whitney U test. Significance levels set at p < 0.05 *, p < 0.01 **, and p < 0.001 ***

Article Snippet: Stereotactic or cannula microinfusion of calcitonin gene‐related peptide receptor antagonist into CeA To block the PBN CGRP neurotransmission to the CeA, we applied the CGRP receptor 1 antagonist, CGRP fragment 8–37 (HY-P0209, MedChemExpress), dissolved in normal saline at 1.8 μg/μl [19], into bilateral CeA with either acute stereotactic microinfusion or intermittent infusion via chronically implanted cannulas.

Techniques: Expressing, Control, Labeling, MANN-WHITNEY

Journal: The journal of headache and pain

Article Title: CGRP-dependent sensitization of PKC-δ positive neurons in central amygdala mediates chronic migraine.

doi: 10.1186/s10194-022-01531-8

Figure Lengend Snippet: Fig. 5 Chemogenetic inhibition of the PKC-δ positive neurons in the CeA. A Schematic illustration of bilateral AAV5-DIO-mCherry (mCherry) or AAV5-DIO-hM4Di-mCherry (hM4Di) injection in the CeA of PKC-δ-Cre mouse. B Schematic illustration of experimental timeline. After 3 weeks of virus expression, PKC-δ-Cre mice were i.p. injected with either vehicle (Ctrl) or NTG (0.1 mg/kg) every other day to day 9 (totally 5 injections) and i.p. injected with CNO (5 mg/kg) on day 10 and 12. The mechanical threshold tested 2 hrs post-treatment after the NTG injection and 1 hr. post-treatment after the CNO injection. C Representative low (up) and high magnification (down) images of mCherry injection sites, confirming the correct injection of viral vectors or controls at bilateral CeA. Scale bar: 100 μm. D Behavioral consequences before and after CNO application PKC-δ-Cre mice pretreated with bilateral hM4Di virus injection in the CeA after chronic vehicle or NTG administration. Sustained mechanical hyperalgesia alleviated 1 hr. after the CNO application on day 10 and 12 (left, n = 4 per group; p < 0.05 *, and p < 0.01 **) while the marble burying test was unaffected (right, n = 4 per group) (E) Sustained basal mechanical hyperalgesia developed after repeated doses of NTG administration in both mCherry and hM4Di group (n = 6 per group). F Behavioral consequences before and after CNO application PKC-δ-Cre mice pretreated with bilateral mCherry and hM4Di virus injection in the CeA and chronic NTG administration. Sustained mechanical hyperalgesia alleviated 1 hr. after the CNO application on day 10 and 12 (left, n = 6 per group; p < 0.01 **, and p < 0.001 ***) while the marble burying test was unaffected (right, n = 6 per group). (G) Representative images of pERK positive neurons in the TG with or without CNO application (left). The numbers of pERK positive neurons in the TG were significantly decreased after the CNO application (right, n = 4 per group; p = 0.009). Scale bar: 100 μm. H The percentage of CGRP/ pERK positive neurons in the TG were significantly decreased after the CNO application (n = 4 per group; p = 0.005). All data shown are mean ± SEM and analyzed by Friedman tests with Dunn’s post hoc test (D, F) or Mann-Whitney U test (G, H). Significance levels set at p < 0.05 *, p < 0.01 **, and p < 0.001 ***

Article Snippet: Stereotactic or cannula microinfusion of calcitonin gene‐related peptide receptor antagonist into CeA To block the PBN CGRP neurotransmission to the CeA, we applied the CGRP receptor 1 antagonist, CGRP fragment 8–37 (HY-P0209, MedChemExpress), dissolved in normal saline at 1.8 μg/μl [19], into bilateral CeA with either acute stereotactic microinfusion or intermittent infusion via chronically implanted cannulas.

Techniques: Inhibition, Injection, Virus, Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis

doi: 10.1038/s41598-019-39170-w

Figure Lengend Snippet: SP and CGRP neutralization reverses DRG-induced EMT and FMT in 11Z cells. ( A ) Representative morphology of 11Z cells treated with medium, the DRG supernatant with pre-treatment with aprepitant (10 −6 M), CGRP fragment 8–37 (10 −6 M), or both aprepitant (10 −6 M) and CGRP 8–37 (10 −6 M), or without for 12 days. Scale bar = 100 μm. ( B ) Left panel: Detection of protein levels of E-cadherin by immunoblotting of lysates of 11Z cells treated with indicated condition for 12 days (n = 3). The grouping of blots from the same protein were not cropped, and all protein blots were from the same gel. Right panel: Relative fold change in protein levels of E-cadherin in 11Z cells treated with indicated condition for 12 days (n = 3). ( C ) Relative fold change of gene expression levels of Snai1, Slug, vimentin, N-cadherin, and PAI-1 in 11Z cells treated with indicated conditions for 12 days (n = 3). Values are normalized to the GAPDH expression levels. ( D ) Results of SP and CGRP neutralization on cellular proliferation of 11Z cells, as measured by CCK-8 assay (n = 8). The 11Z cells were treated with the indicated conditions for 12 days. ( E ) Results of SP and CGRP neutralization on cell migratory capacity, as evaluated by the scratch assay, of 11Z cells treated with indicated conditions for 12 days. The cells were photographed at 0, 12, 24 and 48 hours, respectively, after being scratched. The distance between two edges that cells traversed was calculated relative to the initial scratch distance as measured with pixel values. Scale bar = 100 μm. ( F ) Results of SP and CGRP neutralization on invasiveness. The representative photomicrographs of the invaded 11Z cells in the transwell assay after indicated treatments (Magnification: × 200). Cells, after 12 days’ indicated treatments, were added to the top of transwells coated with Matrigel and treated as indicated. The total number of cells invaded to the bottom of the transwell was then counted. Scale bar = 100 μm. * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001; N: not statistically significant (p > 0.05). Data are represented in means ± SDs. Symbols of statistical significance: *, **, and *** indicate different significant levels when compared with the untreated cells, while #, ## , and ### indicate different significant levels when compared with the cells treated with the DRG supernatant. * or # p < 0.05; ** or ## p < 0.01; *** or ### p < 0.001.

Article Snippet: For inhibitor experiments, cells were pretreated with vehicle or the potent NK1R antagonist aprepitant (Selleckchem) (10 −6 M) or CGRP Fragment 8–37 (CGRP 8–37) (Sigma) (10 −6 M) , a selective competitive antagonist for CGRP receptors, for 1 hour at 37 °C.

Techniques: Neutralization, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Transwell Assay